Non-sputum-based biomarkers for the detection of tuberculosis

/ABSTRACT The global spread of Mycobacterium tuberculosis (Mtb) infection continues to pose a significant public health problem, highlighting the urgent need for simple non-sputum-based point-of-care tests (POCTs) to diagnose active tuberculosis (TB) for use in resource-limited settings, a World Health Organization (WHO) priority. Despite substantial efforts, the only commercially available test (Determine™ TB LAM), which uses polyclonal rabbit IgG to detect Mtb lipoglycan lipoarabinomannan in urine (U-LAM), has very low sensitivity in TB patients without AIDS. Moreover, although studies have shown anti-LAM monoclonal antibodies (mAbs) can improve U-LAM detection, sensitivity with the best-performing mAb pairs remains ≤50% in people with HIV having CD4 cells >200 and those without HIV. Given the need for improved diagnostics to promote early detection and stem the spread of Mtb, our overarching goal is to inform development of a simple, highly sensitive and specific, non- sputum-based POCT(s) for TB. In prior studies, we isolated a series of high-affinity human mAbs that recognize LAM epitopes distinct from those bound by other reported anti-LAM mAbs. Notably, some of our mAbs can detect U-LAM at low levels and in paucibacillary HIV-uninfected TB patients, as well as in lungs of Mtb-infected mice. In this study, we aim to investigate a panel of six mAbs to identify pairs that significantly improve U-LAM detection and accurately detect LAM in exhaled breath concentrate (EBC), which contains LAM concentrations up to 1,000-fold higher than those in urine. We hypothesize that mAbs in our panel will enhance U-LAM detection, bind to EBC-LAM, and enable combined detection of U- and EBC-LAM to achieve the WHO diagnostic TB POCT target sensitivity (≥98% for sputum-smear-positive and ≥65% for smear-negative TB) at a specificity of 95% vs. the TB microbiologic reference standard, irrespective of HIV status. To test these hypotheses, we will use our US sites to enroll patients from diverse TB-endemic regions and use stored TB samples from South Africa and the Democratic Republic of Congo. In Aim 1, we will measure sensitivity of our high-affinity anti-LAM mAbs, identify the best pairs for highly sensitive and specific U-LAM detection, and determine their diagnostic accuracy with urine from TB vs. other respiratory disease patients, stratified by HIV status. In Aim 2, to develop a highly sensitive immunodiagnostic assay for EBC-LAM detection, we will identify mAbs that recognize EBC-LAM, determine the best pairs for EBC-LAM capture and detection, measure TB sensitivity at 95% specificity, and compare sensitivity of combined U- and EBC-LAM detection with that in each fluid alone. Lastly, in Aim 3, we will isolate new mAbs to acylated LAM motifs found in clinical Mtb strains and determine if they show enhanced U- and EBC-LAM detection relative to our existing mAbs. We anticipate these studies will enhance our understanding of the LAM motifs most critical for highly sensitive detection, potentially enabling development of highly sensitive, low-cost U- and EBC-LAM-based TB POCT(s) for improving TB control worldwide. Project Number: 1R01AI185330-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute of Allergy and Infectious Diseases (NIAID) | Principal Investigator: Jacqueline Achkar | Institution: ALBERT EINSTEIN COLLEGE OF MEDICINE, BRONX, NY | Award Amount: $850,711 | Activity Code: R01 | Study Section: Etiology, Diagnostic, Intervention and Treatment of Infectious Diseases Study Section[EDIT] View on NIH RePORTER: https://reporter.nih.gov/project-details/1R01AI18533001A1